Consistent malignant transformation of human breast epithelial cells, or other human cells, in vitro by chemical or physical carcinogens has not been accomplished to date. Among the possible reasons for the failure are 1) the use of inappropriate target cells for transformation studies, i.e. differentiating cells instead of stem cells; and 2) the use of inappropriate laboratory animals as hosts for transformed cells. The major objective of this grant proposal is to examine the validity of these two possibilities. Recently we have observed the existence of a mall subpopulation of human kidney epithelial cells, with stem cell characteristics, i.e., extended life span and capable of contact- insensitive growth on a x-ray lethally irradiated human fibroblast cell mat. This, and our preliminary results, indicate that it is possible to enrich for human breast renewal capacity using the cell mat or an appropriate cell culture medium. Therefore we propose to transform these cells with chemical and physical carcinogens and to select neoplastic transformed cells by using 1) our x-ray lethally irradiated human fibroblast cell mat which, additionally, has been shown to allow the growth of human carcinomas of various origin but not normal cells (except the contact-insensitive putative transformed cells will be characterized biologically and biochemically to demonstrate that they are truly neoplastic transformed. The second major goal of this proposal is to develop new in vivo models capable of accepting and maintaining transformed mammary epithelial cells. The transplantation success rate and growth rate of primary mammary carcinoma (rat and human) and putative in vitro transformed human breast epithelial cells will be examined in 3 strains of immune deficient mice, i.e., conventional athymic nude mice (T-lymphocyte deficient), beige-XID athymic nude mice (T-lymphocyte deficient and LAK lymphocytes). The endocrine system of the immune deficient mice will be altered to provide a hormonal milieu that is rich in human mammotrophic hormones. In this research proposal, we feel that we are addressing and examining two very crucial components of the complex process of ex vivo neoplastic transformation of human breast epithelium, components that have not in the past received sufficient experimental attention.